The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose

Abstract

To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of Arabidopsis was characterized. This mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and affects arabinose-containing pectic cell wall polysaccharides and arabinogalactan proteins. Feeding L-arabinose to mur4 plants restores the cell wall composition to wild-type levels, suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyltransferases. The defect was traced to the conversion of UDP-D-xylose to UDP-L-arabinose in the microsome fraction of leaf material, indicating that mur4 plants are defective in a membrane-bound UDP-D-xylose 4-epimerase.

Figure 1

Pathways involved in l-Ara biosynthesis and reutilization.

Figure 2

Relative Ara content in the cell wall of different organs of wild-type (white bars) and mur4 (black bars) plants. The Ara content is given as a percentage of total neutral cell wall monosaccharides (except Glc). Cotyledons were collected from 200 plants, leaves from 40 plants, 2-cm basal and apical stem segments from 50 plants each, and flowers from 260 plants, and processed in four pools of samples for GC analysis. The bars are the mean of four samples ± sd. For roots, plants were grown on plates and then transferred to hydroponic conditions for 10 d. Roots were collected from 20 plants and processed in three pools; bars are the means of three samples ± sd.

Figure 3

A, Monosaccharide content in leaf-derived cell wall matrix polysaccharides (pectins and hemicelluloses). Identical amounts of cell wall material were used for the wild type (white bars) and mur4 (black bars). The bars represent the mean of three samples ± sd. Total uronic acids determined via the m-hydroxybiphenyl assay were 293 ± 38.7 μg mg⁻¹ dry weight for the wild type (n = 3) and 275 ± 23.0 μg mg⁻¹ dry weight for mur4 (n = 3). B, Glycosyl composition of Arabidopsis AGPs. Identical amounts of total carbohydrates as determined by the phenol-sulfuric acid colorimetric assay were used for wild-type (white bars) and mur4 (black bars) AGP extracts. The bars represent the means of three samples ± sd.

Figure 4

Agarose gel electrophoresis of AGPs from wild-type and mur4 plants. AGPs were extracted from leaf material as soluble polymers from wild-type (A) and mur4 (B) plants and from wild-type (C) and mur4 (D) plants grown in the presence of 50 mm Ara. The AGPs were stained with the Yariv reagent.

Figure 5

Sepharose CL-4B elution profile of the Gal and Ara containing material from AGP extracts of wild-type and mur4 leaves. Each fraction eluting from the Sepharose column was analyzed by GC of alditol acetates. The elution positions of blue dextran (fraction 25, not shown), and the molecular mass markers β-amylase (200 kD), alcohol dehydrogenase (ADH, 150 kD), BSA (66 kD), carbonic anhydrase (CA, 29 kD), and Glc are shown by arrows. Thick solid line, Wild-type ARA; dashed line, wild-type Gal; thin solid line, mur4 Ara; dotted line, mur4 Gal.

Figure 6

Response of wild-type (white bars) and mur4 (black bars) plants to feeding with l-Ara. Plants were grown on agar plates with different concentrations of l-Ara in the medium. Bars represent the means of three samples ± sd.

Figure 7

In vitro assay of the de novo synthesis of UDP-Ara from UDP-GlcUA via UDP-Xyl. Wild-type and mur4 microsomal protein extracts were incubated with radiolabeled UDP-GlcUA. The UDP sugars produced at different time points were hydrolyzed and the sugars were separated and quantified by TLC. Data are mean values ± sd with a sample size of two. ▴, Wild-type Ara; ▵, mur4 Ara; ●, wild-type Xyl; ○, mur4 Xyl.